12alpha-halo-16alpha-hydroxy steroids of the pregnene series



United States Patent 3,047,595 IZoL-HALO-lGu-HYDROXY STEROIDS OF THE PREGNENE SERIES Josef Fried, Princeton, N.J., assignor to Olin Mathieson Chemical Corporation, New York, N.Y., a corporation of Virginia No Drawing. Filed Apr. 14, 1958, Ser. No. 728,051

7 Claims. (Cl. 260397.45)

This application is a continuation-in-part of my parent application Serial No. 677,205, filed August 9, 1957.

This invention relates to, and has for its objects, the provision of new steroids which are useful intermediates in the preparation of physiologically active steroids and to a method of preparing the same.

The new steroids of this invention include steroids of the following general formula and intermediates therefor:

wherein the 1,2-position is saturated or double-bonded, R, R, X and Z are as hereinbefore defined, and Y is hydrogen, hydroxy or acyloxy to the oxygenating action of a microorganism such as Stfeptomyces roseochromogenus, the microbial hydroxylation being carried out by the method described in the US. application of Josef Fried et al., Serial No. 453,411, filed August 31, 1954, now abandoned.

Among the suitable steroids which are utilizable as precursors in the microbial hydroxyla-ti'on can be mentioned 12a-halo-1lp-hydroxyprogesterones (e.g., 12a-fluoro-l1/3-hydroxyprogesterone), 12oz halo-11-ketoprogesterones (e.g., 12a-fiuoro-ll-ketoprogesterone), IZa-halo- A -pregnadiene-llfl-ol-3,2O-diones (e.g., 12a-fluoro-A 'pregnadiene-l1/3-ol-3,20-dione), 12oz haloA -pregnadiene-3,11,20-triones, 12a halo-11B,17adihydroxyprogesterones (e.g., 12ocfluoro-11,8,17a-dihydroxyprogesterone), 12a halo 1l-keto-17a-hydroxyprogesterones, l2a-halo- A -pregnadiene-1lfi,17ot-diol-3,20-diones, 12a haloA pregnadiene-17a-ol-3,11,20-triones, 12m halo-corticosterones (e.g., 12a-fluorocorticosterone), 12a-halo-11-dehydrocorticosterones, 12a haIO-A 'pregnadiene-I113,2l-diol 3,20-diones, 1Zea-halo-A -pregnadiene-21-ol-3,l1,20- triones, 12u-halo-hydrocortisones (e.g., l2a-chlorohydrocortisone and 12a fluorohydrocortisone), 12a halocortisones, 120a -haloprednisolones (e.g., 12a-fluoroprednisolone), 12a-haloprednisones, and 21-este-rs of those steroids which contain a 21-hydroxy group (especially esters with hydrocarbon carboxylic acids of less than ten carbon atoms, as exemplified by the lower alkanoic acids, the lower alkenoic acids, the monocyclic aromatic carboxylic acids, the monocyclic aral kanoic acids, the cycloice alkane carboxylic acids, and the cycloalkene carboxylic acids.

The action of the enzymes of Streptomyces roseochromogenus to produce the 16u-hydroxy derivatives of the steroid precursors can be utilized either by including the steroid in an aerobic culture of the microorganism or by bringing together, in an aqueous medium, the steroid, air and enzymes of non-proliferating cells of the microorganism. In general, the conditions of culturing the Streptomyces roseochromogenus for the purposes of this invention are (except for the inclusion of the steroid to be converted) the same as those of culturing Streptomyces for the production of antibiotics and/ or vitamin B i.e., the microorganism is aerobically grown in contact with (in or on) a suitable fermentation medium. A suitable medium essentially comprises a source of nitrogenous and growth-promoting factors, and an assimilable source of carbon and energy. The latter may be a carbohydrate and/or the steroid itself. Preferably, however, the medium includes an assimilable source of carbon and energy in addition to the steroid; and preferably, also, this source is at least in substantial part a member of the group consisting of (1) fatty acids having at least 14 carbon atoms and (2) fats. Use of such lipid source of carbon and energy (especially use of a fatty oil) is advantageous in that it enhances the availability of the steroid for conversion.

The nitrogen source materials may be organic (e.g., soybean meal, cornsteep liquor, meat extract, and/or distillers solubles) or synthetic (i.e., composed of simple, synthesizable organic or inorganic compounds such as ammonium salts, alkali nitrates, amino acids, urea or thiourea).

As to the energy source material, lipids, especially (1) fatty acids having at least 14 carbon atoms, (2) fats or (3) mixtures thereof, are preferred. Examples of such fats are lard oil, soybean oil, linseed oil, cottonseed oil, peanut oil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin, triolein and trilaurein; and illustrative fatty acids include stearic, palmitic, oleic, linoleic and myristic acids.

Other carbon-containing materials may also be used. For example, such materials as glycerol, glucose, fructose, sucrose, lactose, maltose, dextrins, starches, whey, etc., are adequate carbon source materials. These materials may be used either in purified state or as concentrates, such as whey concentrate, corn, wheat or barley mash; or mixtures of the above may be employed. It is to be noted, however, that the. steroid is added to the fermentation medium essentially as a precursor and not as an energy source.

The fermentation process results in the preparation of a l6a-hydroxy steroid corresponding to the precursor steroid, the 21-ester of the starting steroid, if any, having been hydrolyzed to yield the free 21-hydroxy derivative.

If a l7a-hydroxy steroid is employed as the precursor, the desired final product of this invention, namely, a 1604, 17oc-dihydroxy steroid, is obtained directly. These steroids are useful as disclosed in the parent application, Serial No. 677,205, in the preparation of the corresponding 16a,17 x-acetal or ketal derivatives. If, however, a Nut-unsubstituted steroid is employed as the substrate (Z is hydrogen), then further steps are necessary to convert this steroid to a l7u-hydroxy derivative. This conversion may be eifected according to the next step in the process of this invention by reacting the l7-unsubstituted steroid with a basic reagent, e g., aluminum tertiary butylate, at an elevated temperature, the reaction preferably being conducted in a solvent for the steroid at the reflux temperature of the resulting system. The reaction results in the production of new 16,17-dehydro steroids of this invention having the general formula wherein the 1,2-position is saturated or double-bonded and R, R, X and Y are as hereinbefore defined.

These 16,17-dehydro steroids are then treated with osmium tetroxide, preferably in the presence of an organic base, such as pyridine, to yield the osrnate ester of the corresponding 16a,l7a-dihydroxy derivative, which is then reduced and hydrolyzed, as by treatment with sodium sulflte or hydrogen sulfide, to give the free 16a, 170c-dihydroxy derivative.

Aside from their use as intermediates in the preparation of 16a,17a-acetals and ketals, certain of the new steroids of this invention are also useful per se as physiologically active substances. Thus, those steroids which are unsubstituted in both the 1711- and 2l-positions (Y and Z are hydrogen) possess progestational activity and hence may be used in lieu of known progestational agents, such as progesterone, in the treatment of threatened abortion, dysmenorrhea, etc. Furthermore, those steroids which are unsubstituted in the 17-position and are hydroxylated in the 21-position (Y is hydroxy and Z is hydrogen) possess glucocorticoid activity and hence may be used in lieu of known glucocorticoids, such as hyd-rocortisone, in the treatment of rheumatoid arthritis and the allergic diseases.

The process of this invention can be represented by the following scheme:

CHZY

Soybean meal nutrient gram 15.0 Glucose do 10.0 Soybean oil do 2.2 CaCO do 2.5

Water liter 1 The pH of the medium is adjusted to 7.0101. Fifty ml. portions of the medium are distributed in 250 ml. Erlenmeyer flasks. The flasks are plugged with cotton and sterilized in the usual manner (by autoclaving). When cool, each of the flasks is supplemented with 0.25 ml. of a sterile solution of 12a-fiuorohydrocortisone (I) in dimethyl formamide; thus 0.05% steroid in the medium is provided. Each flask is then inoculated with 5 to 10% of a vegetative inoculum of Streptomyces roseochromogenus (Waksman No. 3689). The inoculated flasks are incubated at 25 with rotatory mechanical shaking for 3 to 4 days, when the contents of 12 flasks are pooled, adjusted in pH to 4:02, and filtered through a clarifying pad on a Buechner funnel. The filtered broth is extracted with four 300-ml. portions of methyl isobutyl ketone, yielding, after removal of the solvent in vacuo, about 200 mg. of crude steroid, which crystallizes on standing. Recrystallization from ethanol yields pure 12afluoro-16a-hydroxyhydrocortisone.

Similarly, 12afluoroprednisolone, 12oc-fluorocortisone, 12a-fluoroprednisone, and 12a-fluoro-11,8,17a-dihydroxyprogesterone yield 12a-fluoro-16ot-hydroxyprednisolone, 12a-fluoro-16ot-hydroxycortisone, 12a-fluoro-16a-hydr0xyprednisone, and 12oc-fluoro-116,16a,17a-trihydroxyprogesterone (VI), respectively.

EXAMPLE 2 12 u-Chloro-I 6 oc-H ydroxycortisone (VIII) Employing the conditions of Example 1, but substituting 0.5 g. of 12a-chlorocortisone (VII) for the 12a-fluorocortisone, there is obtained l2oc-ch1oro-16a-hydroxycortisone.

EXAMPLE 3 12ot-Flu0r0i-11B,16ot,17oc-Trihydr0xyprogesterone (VI) (a) Pre aration of IZ-fluoro-l1B,]6ot-dihydr0xypr0gesterone (IV).-Microbiological hydroxylation of 1200-1111- oro-llfi hydroxyprogesterone (III) with Streptomyces roseochromogenus (Waksman No. 3689) as described in Example 1 produces 16a-hydroxy-12ot-fluoro-1LB-hydroxyprogesterone (IV), which is extracted from the culture filtrate with chloroform. After removal of the solvent in vacuo, the residue is crystallized from acetone-hexane leaving the pure compound having the following properties: Ml. about 218-219; [a] +164 (c., 0.50 in CHCl N25, 240 11111 (e=l6,000); k113i? 2.98, 5.86, 6.03, 6.20

Analysis-Calcd. for C H O F (364.44): C, 69.21; H, 8.02. Found: C, 68.97; H, 7.85.

(17) Preparation of 12a-flu0r0-A -pregnadiene-I15-01- 3,20-a'i0ne (V).A suspension of 400 mg. of 16u-hydroxy-12a-fluoro-1lp-hydroxyprogesterone and 1.2 g. of aluminum tertiary butylate in ml. of anhydrous toluene is heated to reflux for 2 hours. The cooled reaction mixture is washed with dilute hydrochloric acid, water, bicarbonate and again with water until neutral. The organic phase is dried over sodium sulfate and the solvent removed in vacuo. The residual crystalline mass upon recrystallization from acetone-hexane furnishes pure 120cfluoro-A -pregnadiene-11B-ol-3,2O-dione (V) of the following properties: M.P. about 2152l7; [a] +209 (c., 0.56 in chlf), \,l$;, 238 m (e=24,700), A221? 2.94, 5.94, 6.05, 6.16, 6.30;

Analysis--Calcd. for C21H2703F (346.43): C, 72.80; H, 7.86. Found: C, 72.56; H, 7.80.

(0) Preparation of 12wflu0r0-A -pregnene-1]}8,I6a,17atrial-3,20-di0ne (VI).-To a solution of 77 mg. of the diene (V) formed in section (b) and 0.1 ml. of pyridine in 5 ml. of benzene is added 65 mg. of osmium tetroxide. The mixture is allowed to stand in the dark for 18 hours during which period precipitation of a brown crystalline material occurs. For decomposition of the osmate ester, there is added to the mixture 7 ml. of water, 4.6 ml. of methanol, 700 mg. of sodium sulfite and 700 mg. of potassium bicarbonate, and the resulting suspension is stirred for four hours at room temperature. After dilution with 20 ml. of chloroform, the mixture is filtered through Celite and the precipitate washed thoroughly with chloro form. The layers are separated, the chloroform phase washed with water, dried over sodium sulfate and evaporated to dryness in vacuo. The crystalline residue is recrystallized from acetone-hexane, leaving the pure triol (VI) of the following properties: M.P. about 220222, [a] +2 (c., 0.38 in CHCl);

A313 240 m (e=16,200), A231? 2.90, 5.83, 6.02, 6.19;;

EXAMPLE 4 1 Za-Bromo-I 1 5,1 6u-Dihydroxypr0gester0ne (X) Microbiological hydroxylation of 12a-brom0-11p-hy- -droxyprogesterone (IX) with Streptomyces roseochromogenus (Waksman No. 3689) as described in Example 1 produces 16a-hydroxy-12a-bromo-1lfl-hydroxyprogesterone (X) which is extracted from the culture filtrate with chloroform. After removal of the solvent in vacuo, the crystalline residue is recrystallized from acetone-hexane. The pure compound has the following properties: M.P. about 211-212"; [a] -ll01 (in CHCl E32 2.87, 3.01, 5.85, 5.86, 6.16;; Analysis-Oalcd. for C H O Br (425.35): C, 59.29; H, 6.87; Br, 18.79. Found: C, 59.11; H, 7.00; Br, 18.40.

EXAMPLE 5 12a-Br0m0-16a-Hydroxy-1I-Ketoprogesterone (XII Following the procedure of Example 4, 12a-bromo-11- ketoprogesterone (X1) is converted into 16a-hydroxy-12abromo-l l-ketoprogesterone (XII).

The invention may be variously otherwise embodied within the scope of the appended claims.

6 What is claimed is: 1. A compound selected from the group consisting of steroids of the general formulae and wherein R is hydrogen, R is fi-hydroxy and together R and R is keto, X is a halogen of atomic number less than 53, and Z is selected from the group consisting of hydrogen and hydroxy.

2. 12oc-fluoro-1 1,8,16u-dihydroxyprogesterone.

3. IZa-fluoro-l 15,1611,17a-trihydroxyprogesterone.

4. A compound selected from the group consisting of steroids of the general formula and the 1,2-dehydro derivatives thereof, wherein R is hydrogen, R is {B-hydroxy and together R and R is keto, X is a halogen of atomic number less than 35, and Y is selected from the group consisting of hydrogen and hydroxy.

5. 1Zm-haIo-A -pregnadiene-11,8-ol-3,20-dione, wherein the halo has an atomic number less than 5 3.

6. l2a-fiuoro-A -pregnadiene-11/3-ol-3,20-dione.

7. 12u-chloro-16a-hydroxycortisone.

References Cited in the file of this patent UNITED STATES PATENTS 2,822,318 Kroll et a1. Feb. 4, 1958 2,831,003 Thomas Apr. 15, 1958 2,979,517 Herzog Apr. 11, 1961 OTHER REFERENCES McGuckin et al.: 77 J.A.C.S., 1822-24 (1955). 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF STEROIDS OF THE GENERAL FORMULAE AND FIG-01 WHEREIN R IS HYDROGEN, R'' IS B-HYDROXY AND TOGETHER R AND R'' IS KETO, X IS A HALOGEN OF ATOMIC NUMBER LESS THAN 53, AND Z IS SELECTED FROM THE GROUP CONSISTING OF HYDROGEN AND HYDROXY. 